M3 muscarinic receptor-like immunoreactivity in sham operated and obstructed guinea pig bladders.

PURPOSE: Type 3 muscarinic receptors, which are present in the bladder wall, are important for bladder function. However, their role in the context of the urothelium is not well defined. Understanding the role of type 3 muscarinic receptors has been limited by the lack of specific type 3 muscarinic receptor antibodies. Thus, we identified a specific type 3 muscarinic receptor antibody and investigated the site of type 3 muscarinic receptors in sham operated and obstructed guinea pig bladders. MATERIALS AND METHODS: The specificity of 4 commercially available type 3 muscarinic receptor antibodies was determined. Immunohistochemistry was then done in bladder tissue from sham operated and obstructed guinea pig bladders. RESULTS: One of the 4 antibodies examined had the needed specificity in terms of blocking peptide and Western blot characterization. Using this antibody type 3 muscarinic receptor immunoreactivity was associated with muscle cells, nerves and interstitial cells. Four types of interstitial cells were identified, including suburothelial, lamina propria, surface muscle and intramuscular interstitial cells. In the obstructed model the bladder wall was hypertrophied and there was nerve fiber loss. The number of lamina propria, surface muscle and intramuscular interstitial cells was increased but not the number of suburothelial interstitial cells. Also, surface muscle interstitial cells appeared to form clusters or nodes with type 3 muscarinic receptor immunoreactivity. CONCLUSIONS: Nerve loss and the up-regulation of interstitial cells with type 3 muscarinic receptor immunoreactivity may underlie major functional changes in the pathological bladder. This indicates that type 3 muscarinic receptor specific anticholinergic drugs may affect not only the detrusor muscle, as previously thought, but also interstitial cells and nerve fibers.

Nitric oxide signaling modulates synaptic transmission during early postnatal development.

Early γ-aminobutyric acid mediated (GABAergic) synaptic transmission and correlated neuronal activity are fundamental to network formation; however, their regulation during early postnatal development is poorly understood. Nitric oxide (NO) is an important retrograde messenger at glutamatergic synapses, and it was recently shown to play an important role also at GABAergic synapses in the adult brain. The subcellular localization and network effect of this signaling pathway during early development are so far unexplored, but its disruption at this early age is known to lead to profound morphological and functional alterations. Here, we provide functional evidence--using whole-cell recording--that NO signaling modulates not only glutamatergic but also GABAergic synaptic transmission in the mouse hippocampus during the early postnatal period. We identified the precise subcellular localization of key elements of the underlying molecular cascade using immunohistochemistry at the light--and electron microscopic levels. As predicted by these morpho-functional data, multineuron calcium imaging in acute slices revealed that this NO-signaling machinery is involved also in the control of synchronous network activity patterns. We suggest that the retrograde NO-signaling system is ideally suited to fulfill a general presynaptic regulatory role and may effectively fine-tune network activity during early postnatal development, while GABAergic transmission is still depolarizing.

The relationship between prostaglandin E receptor 1 and cyclooxygenase I expression in guinea pig bladder interstitial cells: proposition of a signal propagation system.

PURPOSE: We explored the structural relationship between enzymes producing prostaglandin (cyclooxygenase I) and 1 of the receptor families that respond to prostaglandin (prostaglandin E receptor 1) in the bladder muscle. MATERIALS AND METHODS: Nine male guinea pigs were sacrificed by cervical dislocation. Bladders were removed and fixed in 4% paraformaldehyde in phosphate buffered saline. Frozen sections (10 µm) were cut and stained with antibodies to prostaglandin E receptor type 1, cyclooxygenase I and vimentin. RESULTS: Prostaglandin E receptors 1 was identified on smooth muscle cells, and vimentin positive surface muscle and intramuscular interstitial cells. Muscle staining was less intense than on interstitial cells and had a punctuate appearance. Prostaglandin E receptor 1 expression on interstitial cells was highly localized. Discrete regions of intense staining were noted on interstitial cell processes. Cyclooxygenase I was also expressed in muscle interstitial cells. Cyclooxygenase I positive interstitial cells were more prevalent in the muscle bundles of the inner muscle than in the outer muscle layers. Cyclooxygenase I staining was noted on discrete regions of the cell or cell processes. Double staining with prostaglandin E receptor 1 and cyclooxygenase I suggested that cell regions expressing the former are different from those expressing the latter. CONCLUSIONS: The discovered arrangement of prostaglandin E receptor 1 and cyclooxygenase I may have the potential to facilitate the propagation of signals in the interstitial cell network. Such a signaling system may have a role in coordinating events, as in bladder pathology, facilitating the global coordinated changes associated with bladder wall remodeling.

Prostaglandin receptor EP1 and EP2 site in guinea pig bladder urothelium and lamina propria.

PURPOSE: Urothelium has 2 main functions. It is a barrier to urine and has a sensory role. In response to stretch urothelium releases various substances that modulate afferent nerve activity. Recent data on the localization of cyclooxygenase type 1, the enzyme responsible for prostaglandin production, suggests that prostaglandin may have complex local action. MATERIALS AND METHODS: The bladders of 7 guinea pigs were stained for prostaglandin receptors type 1 and 2, and costained for vimentin and cyclooxygenase I. RESULTS: Prostaglandin receptor type 1 staining was seen in urothelial cells and in the suburothelium. Urothelial staining, which was often punctuate and weak, was detected in all urothelial cell layers, including suburothelial cells. In contrast, strong prostaglandin receptor type 2 staining was seen in the urothelium and in suburothelial cells. Cyclooxygenase I was absent in interstitial cells and umbrella cells with the highest concentration in the basal cell layer. CONCLUSIONS: Interstitial cells express prostaglandin receptor types 1 and 2, indicating that they can respond to prostaglandin. Umbrella cells do not express cyclooxygenase I. Cyclooxygenase I was present in basal urothelial cells, making them a possible site of prostaglandin synthesis. Thus, prostaglandin produced by urothelium may target prostaglandin receptor types 1 and 2 in the urothelium and suburothelium. Therefore prostaglandin is hypothesized to have a role in signal regulation in the bladder wall.

Stretch independent regulation of prostaglandin E(2) production within the isolated guinea-pig lamina propria.

OBJECTIVE: To use an isolated preparation of the guinea-pig bladder lamina propria (LP) to investigate the effects of adenosine tri-phosphate (ATP) and nitric oxide (NO) on the release of prostaglandin E(2) (PGE(2)). MATERIALS AND METHODS: The bladders of female guinea-pigs (200-400 g) were isolated and opened to expose the urothelial surface. The LP was dissected free of the underlying detrusor muscle and cut into strips from the dome to base. Strips were then incubated in Krebs buffer at 37 degrees C. Each tissue piece was then exposed to the stable ATP analogue, BzATP, and a NO donor, diethylamine-NONOate (DEANO), and the effect on PGE(2) output into the supernatant determined using the Parameter(TM) PGE(2) enzyme immunoassay kit (R & D Systems, Abingdon, UK). Experiments were repeated in the presence of purinergic receptor and cyclooxygenase (COX) enzymes, COX I and COX II, antagonists. The cellular location of COX I, COX II and neuronal NO synthase (nNOS) within the bladder LP was also determined by immunohistochemistry. RESULTS: PGE(2) production was significantly increased by BzATP. Antagonist studies showed the purinergic stimulation involved both P(2)X and P(2)Y receptors. The BzATP response was inhibited by the COX inhibitor indomethacin (COX I >COX II) but not by DUP 697 (COX II >COX I). Thus, BzATP stimulation occurs because of COX I stimulation. NO had no effect on PGE(2) production over the initial 10 min of an exposure. However, PGE(2) output was increased 100 min after exposure to the NO donor. In the presence of NO, the BzATP stimulation was abolished. Immunohistochemistry was used to confirm the location of COX I to the basal and inner intermediate urothelial layers and to cells within the diffuse layer of LP interstitial cells. In addition, nNOS was also located in the basal urothelial layers whilst COX II was found in the interstitial cell layers. CONCLUSIONS: There is complex interaction between ATP and NO to modulate PGE(2) release from the bladder LP in the un-stretched preparation. Such interactions suggest a complex interrelationship of signals derived from this region of the bladder wall. The importance of these interactions in relation to the physiology of the LP remains to be determined.

On the origins of the sensory output from the bladder: the concept of afferent noise.

For many people a recurrent strong desire to void, sometimes with incontinence, diminishes their quality of life. At present there are few insights into what underlies these problems. The condition is described as the 'overactive bladder symptom complex' but this definition is proving to be unhelpful. It focuses on overt bladder contractions rather than the main problem, which is altered and heightened sensation. Also, current approaches that describe bladder sensations as episodic and leading to voiding as 'first and second sensation to void' might also be misleading if they are taken too literally and used to suggest mechanisms. Current research is beginning to focus on the mechanisms that generate afferent information from the bladder and how it can become altered. As these views develop it is crucial that we appreciate the diversity of the bladder afferent system and distinguish between afferent and sensory information; in this review we explore this underlying complexity. The central nervous system (CNS) receives vast amounts of information from the bladder, which arises from different locations, uses different fibre types and involves different methods. The CNS is continually being bombarded with 'afferent noise'. The challenge now is to understand the nature and components of this 'afferent noise' and which components are essential to sensation. The emerging picture is complex, but this complexity must not be negated or oversimplified. It must be embraced and incorporated it into thinking when designing experiments, analysing data, diagnosing patients and evaluating treatment.

Ubiquitin hydrolase (protein gene product 9.5) in the obstructed bladder: evidence for tissue remodelling involving a subset of interstitial cells.

OBJECTIVE: To examine the expression of ubiquitin hydrolase (UH), an enzyme which is part of the ubiquitin-proteasome system involved in the regulation of cell growth and differentiation, to gain an insight into the cell types and processes underlying the tissue remodelling that occur after bladder neck damage. MATERIALS AND METHODS: Three groups of male guinea pigs were used, comprising controls (not operated, four), sham (five) and obstructed (six). The bladder outlet was obstructed by implanting a silver ring around the urethra, which was left in situ for 2-4 weeks. Sham-operated guinea pigs had the same operative procedure but no ring was implanted. The bladders were removed and incubated in Krebs' solution at 36 degrees C, gassed with 95% O2 and 5% CO2, Tissues were then fixed in 4% depolymerized paraformaldehyde and processed for immunohistochemistry. We used antibodies raised against UH, cyclooxygenase type I and vimentin. Specific antibody binding was visualized using the appropriate secondary antibodies. RESULTS: Staining with an antibody to UH showed the presence of both sensory and motor nerves in control, sham and obstructed bladders. In the control bladders this was the predominant staining pattern. In the sham and obstructed bladders UH staining revealed additional positive cell types; cells associated with the outermost layers of the urothelium (the umbrella cells), in the lamina propria (the lamina propria interstitial cells (lp-ICs), on the surface of the muscle bundles (surface muscle, sm-ICs) and on the serosal surface (muscle coat, mc-ICs). All ICs stained with vimentin. The ICs within the muscle bundles (intramuscular, im-ICs) did not stain with UH. The number and density of the UH-positive cells was greater in the obstructed than in the sham bladders, suggesting a change in relation to the severity of damage to the bladder neck. CONCLUSION: The expression of UH implies the re-targeting of proteins marked for degradation in the proteasome. Increased expression of UH in the lp-ICs and sm-ICs shows that these cells are active in the early and late stages of the tissue remodelling resulting from obstruction. These results show a further subset of ICs that might be involved in the increased deposition of extracellular material and tissue remodelling.

M(3) muscarinic receptor expression on suburothelial interstitial cells.

OBJECTIVE: To identify the cells expressing the M(3) muscarinic receptor subtype in the lamina propria of the bladder. MATERIALS AND METHODS: The bladders from five normal guinea pigs were isolated and fixed in 4% paraformaldehyde. Tissues sections (10 microm) were then cut and stained with antibodies to the type 3 muscarinic receptor (M(3)), the interstitial cell marker vimentin and the nonspecific nerve marker PGP 9.5. The specificity of the antibody to the M(3) receptor was established using the complementary blocking peptide and Western blot analysis of human embryonic kidney (HEK) cells transfected to express the M(3) receptor protein. RESULTS The M(3) antibody pre-incubated with its blocking peptide showed no immunohistochemical staining. Investigating this antibody using HEK cells transfected to express the M(3) receptor protein and control HEK cells showed a single band in the transfected cells and no band in the control cells. M(3) receptor immunoreactivity (M(3)-IR) was detected primarily on a dense network of vimentin-positive (vim(+)) cells lying immediately below the urothelium, i.e. the suburothelial interstitial cells (Su-ICs). The M(3)-IR was punctate and appeared to be located on the cell surface. The diffuse network of cells in the remaining regions of the lamina propria showed no M(3)-IR. Few nerve fibres were associated with the M(3)-IR Su-ICs. The M(3)-IR Su-ICs were most numerous and prominent in the lateral wall. The number of M(3)-IR/vim(+) cells diminished towards the bladder base and were absent in the bladder urethral junction. In the base and urethral junction there were vim(+) cells that were not M(3)-IR. A population of umbrella cells in the lateral wall also showed weak punctate M(3)-IR. CONCLUSIONS Using a well-characterized M(3) antibody, these results show for the first time that the M(3) muscarinic receptor in the lamina propria is located specifically on the Su-ICs. The physiological role of these cells is unknown and consequently the significance of what appears to be a cholinergic signalling system is unclear. Previously published data showed that these cells respond to nitric oxide and atrial natriuretic peptide with an increase in cGMP and possibly prostaglandin. All of these observations, taken together, suggest that the Su-ICs receive multiple inputs and that they must be part of a complex signalling system in this region of the bladder wall.

Thyroid hormone level positively regulates NOS and cGMP in the developing rat cerebellum.

Thyroid hormones and nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling play a significant role in the structural development of the cerebellum, respectively. In the present study, the possible contribution of neuronal NO synthase (NOS) and cGMP in the thyroid hormone-induced structural changes was investigated in the cerebella of postnatal rats at different hormone levels. Animals were treated from postnatal day 4 until days 7, 14, and 21, by i.p. injection of 1 microg thyroxine (T(4))/10 g body weight/4 days, or p.o. with 100 microg 6-n-propyl-2-thyouracil (PTU)/10 g body weight/day. Control groups consisted of i.p. and p.o. vehicle controls and PTU/T(4)-treated animals. Measurement of serum fT(4), TSH as well as total T(3) and T(4) concentration of the cerebellar tissue indicated the changed thyroid status. nNOS extensively expressed in growing parallel fibers revealed by quantitative Western blot and layer analysis of immunohistochemically labeled coronal sections. Simultaneously, the cGMP concentration increased and the distribution of cGMP-immunoreactive (cGMP-IR) material in Purkinje cell perikarya and in the molecular layer expanded during cerebellar development. T(4) increased nNOS and cGMP level, and accelerated the development of nNOS-IR parallel fibers and cGMP-IR molecular layer. In contrast, PTU retarded the development by decreasing nNOS and cGMP concentration and slowing down layer formation. A single dose of T(4) could rescue the PTU-induced changes. Results suggest that the contribution of nNOS- NO/cGMP signaling in thyroid hormone regulated structural maturation of the cerebellum. The possible involvement of neurotrophins, calcium, and apoptotic events in this process was discussed.

Elevated glutamate and NMDA disrupt production of the second messenger cyclic GMP in the early postnatal mouse cortex.

The second messenger cyclic guanosine monophosphate (cGMP) plays many roles during nervous system development. Consequently, cGMP production shows complex patterns of regulation throughout early development. Elevated glutamate levels are known to increase cGMP levels in the mature nervous system. A number of clinical conditions including ischemia and perinatal asphyxia can result in elevated glutamate levels in the developing brain. To investigate the effects of elevated glutamate levels on cGMP in the developing cortex we exposed mouse brain slices to glutamate or N-methyl D-aspartate (NMDA). We find that at early postnatal stages when the endogenous production of cGMP is high, glutamate or NMDA exposure results in a significant lowering of the overall production of cGMP in the cortex, unlike the situation in the mature brain. However, this response pattern is complex with regional and cell-type specific exceptions to the overall lowered cGMP production. These data emphasize that the response of the developing brain to physiological disturbances can be different from that of the mature brain, and must be considered in the context of the developmental events occurring at the time of disturbance.

Regional differences in sensory innervation and suburothelial interstitial cells in the bladder neck and urethra.

OBJECTIVE: To identify and characterize possible structural specialisations in the wall of the lower urinary tract (LUT) in the region of the bladder urethral junction (BUJ), with the specific objective of identifying regional variations in sensory nerve fibres and interstitial cells (ICs). MATERIALS AND METHODS: The bladder base and urethra was removed from five male guinea pigs killed by cervical dislocation. Tissue pieces were incubated in Krebs' solution at 36 degrees C, gassed with 95% O(2) and 5% CO(2), fixed in 4% paraformaldehyde and processed for immunohistochemistry. The nonspecific marker vimentin and the general neuronal marker protein gene product (PGP) 9.5 were used to identify ICs and nerve fibres, respectively. Specific antibody binding was visualized using the appropriate secondary antibodies. RESULTS: The wall of the LUT in the region immediately between the bladder base and the urethra, the BUJ, differed in its cellular composition relative to the adjacent areas. PGP-positive (PGP(+)) nerve fibres, presumptive afferent fibres, lay within the urothelium running between the epithelial cells. There were two general nerve patterns: branching fibres with no varicosities, and complex fibres with varicosities. Fibre collaterals with varicosities exited the urothelium and occupied the space under the urothelium adjacent to the layer of suburothelial ICs. The latter, lamina propria and around the muscle bundles were identified using vimentin (vim(+)). In the base a few vim(+) cells were also PGP(+). In the region of the BUJ there was a decrease in the amount of smooth muscle. In this region, below the lamina propria, there was an area densely populated with vim(+)/PGP(+) ICs. Nerve fibres ran between the cells in this region. CONCLUSION: These structural specialisations within the urothelium and deeper layers of the BUJ suggest that they might be associated with specific functions. The localized highly branched network of the putative afferent nerves suggests the presence of a local axonal reflexes involving possible cross-talk between the urothelium and suburothelial layer. The function of the specialized region of ICs is not known and must await further information on the functional properties of this novel cell type. These observations show further the cellular heterogeneity of the cells in the LUT and the complexity of the structures. One of the major current challenges in functional urology is to understand the relationships between these novel structures and overall bladder and urethral function.

Two autocrine pathways to regulate cyclic GMP synthesis in cultured human retinal pigment epithelial cells.

AIM: To investigate the role of two separate enzymatic pathways [soluble (sGC) vs. particulate (pGC) guanylyl cyclase] in the synthesis of cyclic GMP (cGMP) in cultured human retinal pigment epithelial (RPE) cells. METHODS: cGMP accumulation was evaluated by quantitative analysis of cGMP immunoreactivity. RPE cells were also stained for inducible nitric oxide synthase (iNOS), ANP and beta(1)- and alpha(2)-subunits of sGC. RESULTS: We showed nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and iNOS immunoreactivity in RPE cells. Incubation of the cells in the presence of 1 mM IBMX to inhibit phosphodiesterase activity, and the simultaneous inhibition of NOS activity with L-NAME suggested the involvement of sGC in maintaining a low level of cGMP in the RPE cells. The involvement of sGC was further supported by detection of the beta(1)- and alpha(2)-subunits of sGC. Incubation of the cells in the presence of atrial natriuretic peptide (ANP) to stimulate pGC strongly increased cGMP immunoreactivity. We also demonstrated the presence of ANP in all RPE cells. CONCLUSION: Cultured human RPE cells are capable of producing cGMP after stimulation of sGC or pGC. The presence of iNOS and ANP in all cells suggests two different autocrine pathways of stimulating cGMP production in these cells. The possible role of cGMP in the regulation iNOS gene expression and in the regulation of ANP is discussed.

Cyclic GMP in the pig vitreous and retina after experimental retinal detachment.

PURPOSE: Earlier studies have revealed a decreased level of cGMP in vitreous fluid obtained from patients with a retinal detachment. To further investigate this phenomenon, we developed an experimental retinal detachment model in pigs. METHODS: Experimental unilateral retinal detachments were induced in pig eyes by subretinal injection of 0.25% sodium hyaluronate. Fourteen days later the vitreous and retinas were analyzed for cGMP expression. Following enucleation, the retinas were incubated in the presence of a nonselective phosphodiesterase inhibitor (IBMX), and the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). cGMP was visualized in retinal wholemounts by immunochemistry combined with a computer based stereology system. cGMP levels in vitreous were determined by ELISA. RESULTS: The mean vitreous cGMP level in pig eyes with a retinal detachment (1.45 pmol/ml) was significantly lower compared to the mean level of cGMP in healthy pig eyes (4.61 pmol/ml; p=0.028 was considered significant). In the inner retina, ANP as well as SNP induced cGMP immunoreactivity in both detached and healthy retinas. After incubation with ANP, cGMP could also be detected in the outer nuclear layer of the detached retina, whereas this was not the case in the normal retina. CONCLUSIONS: Experimental retinal detachment in the pig eye leads to a decrease of cGMP levels in vitreous similar to that observed in clinical studies. This model may be helpful to analyze the mechanisms involved in cGMP dynamics following retinal detachment.

Human apolipoprotein C-I expression in mice impairs learning and memory functions.

The H2 allele of APOC1, giving rise to increased gene expression of apolipoprotein C-I (apoC-I), is in genetic disequilibrium with the APOE4 allele and may provide a major risk factor for Alzheimer's disease (AD). We found that apoC-I protein is present in astrocytes and endothelial cells within hippocampal regions in both human control and AD brains. Interestingly, apoC-I colocalized with beta-amyloid (Abeta) in plaques in AD brains, and in vitro experiments revealed that aggregation of Abeta was delayed in the presence of apoC-I. Moreover, apoC-I was found to exacerbate the soluble Abeta oligomer-induced neuronal death. To establish a potential role for apoC-I in cognitive functions, we used human (h) APOC1(+/0) transgenic mice that express APOC1 mRNA throughout their brains and apoC-I protein in astrocytes and endothelial cells. The hAPOC1(+/0) mice displayed impaired hippocampal-dependent learning and memory functions compared with their wild-type littermates, as judged from their performance in the object recognition task (P = 0.012) and in the Morris water maze task (P = 0.010). ApoC-I may affect learning as a result of its inhibitory properties toward apoE-dependent lipid metabolism. However, no differences in brain mRNA or protein levels of endogenous apoE were detected between transgenic and wild-type mice. In conclusion, human apoC-I expression impairs cognitive functions in mice independent of apoE expression, which supports the potential of a modulatory role for apoC-I during the development of AD.

Nitrotyrosine in human neonatal spinal cord after perinatal asphyxia.

BACKGROUND: Spinal cord injury has been reported after perinatal asphyxia in full-term neonates. OBJECTIVES: To examine the role of excessive nitric oxide production in perinatal spinal cord injury. SUBJECTS AND METHODS: Tissue samples of 18 full-term neonates who died of hypoxic-ischemic encephalopathy were analyzed for the presence of nitrotyrosine (NT). RESULTS: NT was demonstrated in 5 of these 18 neonates. In addition, activated caspase 3, a marker of apoptosis, and CD68, as a marker of inflammation, could be demonstrated in some infants. CONCLUSIONS: excessive nitric oxide production and subsequent NT formation is seen in spinal cord tissue after severe perinatal asphyxia. This finding may be relevant for the development of neuroprotective strategies.

Involvement of nitric oxide in depolarization-induced suppression of inhibition in hippocampal pyramidal cells during activation of cholinergic receptors.

Several types of neurons are able to regulate their synaptic inputs via releasing retrograde signal molecules, such as endocannabinoids or nitric oxide (NO). Here we show that, during activation of cholinergic receptors, retrograde signaling by NO controls CB1 cannabinoid receptor (CB1R)-dependent depolarization-induced suppression of inhibition (DSI). Spontaneously occurring IPSCs were recorded in CA1 pyramidal neurons in the presence of carbachol, and DSI was induced by a 1-s-long depolarization step. We found that, in addition to the inhibition of CB1Rs, blocking the NO signaling pathway at various points also disrupted DSI. Inhibitors of NO synthase (NOS) or NO-sensitive guanylyl cyclase (NO-sGC) diminished DSI, whereas a cGMP analog or an NO donor inhibited IPSCs and partially occluded DSI in a CB1R-dependent manner. Furthermore, an NO scavenger applied extracellularly or postsynaptically also decreased DSI, whereas L-arginine, the precursor for NO, prolonged it. DSI of electrically evoked IPSCs was also blocked by an inhibitor of NOS in the presence, but not in the absence, of carbachol. In line with our electrophysiological data, double immunohistochemical staining revealed an NO-donor-induced cGMP accumulation in CB1R-positive axon terminals. Using electron microscopy, we demonstrated the postsynaptic localization of neuronal NOS at symmetrical synapses formed by CB1R-positive axon terminals on pyramidal cell bodies, whereas NO-sGC was found in the presynaptic terminals. These electrophysiological and anatomical results in the hippocampus suggest that NO is involved in depolarization-induced CB1R-mediated suppression of IPSCs as a retrograde signal molecule and that operation of this cascade is conditional on cholinergic receptor activation.

Damage to the bladder neck alters autonomous activity and its sensitivity to cholinergic agonists.

OBJECTIVE: To identify and describe changes to the motor component of the motor/sensory system, which contributes to sensation during the filling phase of the micturition cycle, as a result of surgically induced bladder pathology, i.e. damage to the bladder neck and outlet obstruction. MATERIALS AND METHODS: Adult male guinea pigs (294-454 g) were assigned initially into three groups: (i) normal guinea pigs with no surgical intervention (control, seven); (ii) guinea pigs which, with full surgical anaesthesia, had a silver ring implanted around the bladder neck (obstructed, 13); and (iii) guinea pigs operated to expose the bladder neck but with no implantation of a ring (sham, six). At 2-4 weeks after surgery the bladders were isolated, weighed and the pressure recordings used to identify autonomous activity. RESULTS: The bladder weights in all operated groups, including the sham, were greater than controls. Bladder weights in the obstructed guinea pigs varied considerably, reflecting the degree of pathological change. Consequently, bladders from this group were divided into those with high (OBH) and those with low bladder weight (OBL). The mean (sd) amplitudes of the autonomous contractions were 1.1 (0.1), 10.8 (1.8), 11.4 (2.5) and 17.1 (4.0) cmH(2)O in control, sham, OBL and OBH bladders, respectively, indicating a progressive alteration in function with the pathology. The changes in the sham group suggested that the pathological changes were not the result of obstruction but damage to the bladder neck, the implantation of the silver rings exacerbating the damage. There were episodes of rapid phasic activity (bursts) in 10 of 13 of the ring-implanted bladders, and in two of six in the sham group, but never in controls. Neither the autonomous activity nor the bursts were affected by tetrodotoxin (1 microm) or atropine (3 microm) but they were abolished by noradrenaline (3 microm). In control bladders, adding the muscarinic agonist arecaidine produced a transient acceleration of phasic activity and increased the amplitude of the contractions. There was a similar acceleration of activity in all the operated groups but the concentrations needed to achieve an increase in frequency were significantly lower, the relative sensitivity to arecaidine being OBH >/= OBL > sham > control. CONCLUSION: The mechanism involved in controlling the frequency of the motor component of the motor/sensory system, the 'pacemaker', appears to become progressively 'supersensitive' to cholinergic stimulation with the development of pathology. These observations are discussed in relation to the motor/sensory system and the origins of sensation in the bladder. The argument is proposed that damage to the bladder neck, not obstruction per se, results in altered nonmicturition activity which contributes to increased afferent output. In turn this contributes to the increased sensations of urge associated with bladder dysfunction. The cholinergic regulation of this altered 'pacemaker' might be the target for one of the therapeutic actions of anticholinergic drugs.

The effects of exogenous prostaglandins and the identification of constitutive cyclooxygenase I and II immunoreactivity in the normal guinea pig bladder.

OBJECTIVES: To establish the functional consequences of exposing the isolated whole bladder preparation to exogenous prostaglandins (PGE(1), PGE(2), PGF(2alpha)) and to determine which cells express cyclooxygenase (COX) types I and II, to generate PG to effect these changes in vivo. MATERIALS AND METHODS: Fifteen female guinea pigs (270-350 g) were used, i.e. seven for structural studies and eight for physiological measurement. For the structural study pieces of the lateral wall were incubated separately in Krebs' solution at 36 degrees C, gassed with 95% O(2) and 5% CO(2) with 1 mm isobutyl-methyl-xanthene. Individual pieces were then exposed to 100 microm of the nitric oxide (NO) donor NONOate for 10 min; control tissues remained in Krebs' solution. Tissues were then fixed in 4% paraformaldehyde. For the physiological experiments bladders were isolated and a cannula inserted into the urethra to monitor intravesical pressure. The bladders were suspended in a chamber containing carboxygenated physiological solution at 33-36 degrees C. All drugs were added to the abluminal bladder surface. RESULTS: In the resting bladder there were small spontaneous transient rises in pressure, i.e. autonomous activity. Exposure to PGE(2) (3-300 nM) resulted in an increase in basal pressure on which were superimposed autonomous activity, which was increased both in amplitude and frequency. The changes in the amplitude and frequency depended on the concentration of PGE(2). After a brief exposure (240 s) to PGE(2) the augmentation of the autonomous activity continued for >60 min despite regular washing. The responses were similar with PGE(1) but the responses to PGF(2alpha) and arachidonic acid were reduced. The augmented activity was reduced by the EP1/EP2 receptor blocking agent AH6809 (10 microm). Using an antibody to the 70 kDa constitutive form (COX I), COX I immunoreactivity (COX I-IR) was located in cells in the basal urothelium, in lamina propria and cells on the surface of the inner muscle bundles. There were few COX I-IR cells associated with the outer muscle bundles. The COX I-IR cells lying within the lamina propria were distinct from the suburothelial cells which respond to NO with an increase in cGMP. The lamina propria COX I-IR cells appeared to form a network surrounding muscle trabeculae within the inner muscle layer. COX II-IR was associated with the nuclei of cells in the urothelium, lamina propria and muscle. CONCLUSIONS: These data show that PGs regulate autonomous activity. Potential sources of endogenous PG were identified. It is unclear how the PGs produced by these cells alter autonomous activity. There might be a direct activation of the muscle by PGs released by the network of superficial muscle interstitial cells, or PG released from the urothelium might influence phasic contractile activity via networks of COX I-IR interstitial cells. The possible roles and importance of this mechanism for bladder physiology and pathology are discussed.

Expression of the cGMP-specific phosphodiesterases 2 and 9 in normal and Alzheimer's disease human brains.

We studied the mRNA expression of cGMP-hydrolysing phosphodiesterases (PDEs) in selected brain areas of normal elderly people and patients with Alzheimer's disease. Using radioactive in-situ hybridization histochemistry we found a widespread distribution of the mRNA for PDE2 and PDE9, whereas no specific hybridization signal was observed for PDE5. We observed PDE2 and PDE9 mRNA in all cortical areas studied (insular cortex, entorhinal cortex and visual cortex), although to a different extent. PDE2 mRNA was high in the claustrum, whereas PDE9 mRNA was moderate. PDE2 and PDE9 mRNAs was present in the putamen. No cGMP-hydrolysing PDE expression was observed in the globus pallidus. PDE2 and PDE9 mRNA was observed in all subareas of the hippocampus; however, there were significant differences in the amount of expression. In the Purkinje and cerebellar granule cells only PDE9 expression was observed. PDE2 and PDE9 mRNA expression was not significantly different in Alzheimer's disease brains.

Cyclic GMP synthesis by human retinal pigment epithelial cells is mainly mediated via the particulate guanylyl cyclase pathway.

BACKGROUND/AIMS: Cyclic 3',5'-guanosine monophosphate (cGMP), a central molecule in the phototransduction cascade, is also involved in a number of other physiological processes in the retina, like stimulating the absorption of subretinal fluid by activating the retinal pigment epithelium (RPE) cell pump. The aim of this study was to quantify cGMP synthesis by RPE cells and to investigate the role of two separate enzymatic pathways (soluble versus particulate guanylyl cyclase) in its production. METHODS: cGMP expression was evaluated by immunochemistry and radioimmunoassay following culture of the D407 RPE cell line in the presence of a nonselective phosphodiesterase inhibitor (IBMX), in combination with the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). RESULTS: Stimulation of the particulate guanylyl cyclase in RPE cells with ANP resulted in high intra- and extracellular cGMP levels. Stimulation of the soluble guanylyl cyclase by SNP resulted in a slight elevation of cGMP levels compared to controls. CONCLUSIONS: These results show that cultured human RPE cells are capable of producing cGMP and that most cGMP is generated following stimulation of the particulate guanylyl cyclase pathway.